Human FGF-20 gene and gene expression products

ABSTRACT

This invention relates to human fibroblast growth factor (hFGF-20), and to variants thereof and to polynucleotides encoding FGF-20. The invention also relates to diagnostic and therapeutic agents related to the polynucleotides and proteins, including probes and antibodies, to methods of treating neuronal degenerative disease such as Parkinson&#39;s disease and to methods of treating disorders of the cochlea including those causing hearing loss. The invention also relates to rat fibroblast growth factor (rFGF-20), and to variants thereof and polynucleotides encoding rFGF-20.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/639,117 filedAug. 12, 2003, which is a divisional application of U.S. Ser. No.09/692,945 filed Oct. 20, 2000, and claims priority from U.S. PatentApplication No. 60/161,162 filed Oct. 22, 1999, and U.S. PatentApplication No. 60/187,856 filed Mar. 8, 2000, which are incorporated byreference herein in their entirety.

TECHNICAL FIELD

The present invention relates to nucleic acid sequences encoding membersof the fibroblast growth factor (FGF) family, and to polypeptidesencoded by the nucleic acid sequences.

BACKGROUND OF THE INVENTION

The substantia nigra is an area of the brain that has generatedintensive research. Interest in the substantia nigra was originallybased on the finding that degeneration of dopaminergic neurons in thearea causes Parkinson's disease. In addition, the substantia nigra hasbeen strongly implicated in thought and affective disorders (1).Therefore, neurotrophic factors for dopaminergic neurons in thesubstantia nigra are of substantial clinical interest.

Glial cell line-derived neurotrophic factor (GDNF) is the firstneurotrophic factor documented to enhance the survival of midbraindopaminergic neurons (Lin, L.-F. H. et al., Science 260:1130-1132(1993)). Persephin, Artemin, BDGF and NT-3 also enhance the survival ofmidbrain dopaminergic neurons and have clinical potential in thetreatment of Parkinson's disease (Milbrandt, J. et al., Neuron20:245-253 (1998); Baloh, R. H. et al., Neuron 21:1291-1302 (1998);Hyman, C. et al., Nature 350:230-232 (1991); Hyman, C. et al., J.Neurosci. 14:335-347 (1994)). However, GDNF was reported to be widelyexpressed in neurons of the brain (Pochon, N. A. et al., Eur. J.Neurosci. 9:463-471 (1997)). Persephin was also widely expressed inseveral major tissues including heart, kidney, liver and brain(Milbrandt, J. et al., Neuron 20:245-253 (1998)). Artemin in brain wasexpressed in the basal ganglia and thalamus, suggesting that itinfluences the subcortical motor system (Baloh, R. H. et al., Neuron21:1291-1302 (1998)). BDNF and NT-3 were predominantly expressed in thehippocampus (Ernfors, P. et al., Neuron 5:511-526 (1990)). Therefore,these neurotrophic factors appear not to be specific for dopaminergicneurons in the substantia nigra.

The prototypic fibroblast growth factors (FGFs), FGF-1 (aFGF) and FGF-2(bFGF), were originally isolated from brain and pituitary as mitogensfor fibroblasts. However, FGF-1 and FGF-2 are widely expressed indeveloping and adult tissues, and are polypeptides with multiplebiological activities including angiogenesis, mitogenesis, cellulardifferentiation and repair of tissue injury (Baird, A. et al., CancerCells 3:239-243 (1991); Burgess, W. H. et al., Annu. Rev. Biochem.58:575-606 (1989)). According to the published literature, the FGFfamily now consists of at least nineteen members, FGF-1 to FGF-19(Dickson, C. et al., Ann. NY Acad. Sci. 638:18-26 (1991); Yoshida, T. etal., Ann. NY Acad. Sci. 638:27-37 (1991); Goldfarb, M. et al., Ann. NYAcad. Sci. 638:38-52 (1991); Coulier, F. et al., Ann. NY Acad. Sci.638:53-61 (1991); Aaronson, S. A. et al., Ann. NY Acad. Sci. 638:62-77(1991); Tanaka, A. et al., Proc. Natl. Acad. Sci. USA 89:8928-8932(1992); Miyamoto, M. et al., Mol. Cell. Biol. 13:4251-4259 (1993);Yamasaki, M. et al., J. Biol. Chem. 271:15918-15921 (1996); Smallwood,P. M. et al., Proc. Natl. Acad. Sci. USA 93:9850-9857 (1996); McWhirter,J. R. et al., Development 124:3221-3232 (1997); Miyake, A. et al.,Biochem. Biophys. Res. Commun. 243:148-152 (1998); Hoshikawa, M. et al.,Biochem. Biophys. Res. Commun. 244:187-191 (1998); Ohbayashi, N. et al.,J. Biol. Chem. 273:18161-18164 (1998); Nishimura, T. et al., Biochim.Biophys. Acta 1444:148-151 (1999)). FGF-3 was identified to be a commontarget for activation by the mouse mammary tumor virus (Dickson, C. etal., Ann. NY Acad. Sci. 638:18-26 (1991)). FGF-4 to FGF-6 wereidentified as oncogene products (Yoshida, T. et al., Ann. NY Acad. Sci.638:27-37 (1991); Goldfarb, M. et al., Ann. NY Acad. Sci. 638:38-52(1991); Coulier, F. et al., Ann. NY Acad. Sci. 638:53-61 (1991)). FGF-10was identified from rat lung by homology-based polymerase chain reaction(PCR) (Yamasaki, M. et al., J. Biol. Chem. 271:15918-15921 (1996)).FGF-11 to FGF-14 (FGF homologous factors (FHFs) 1 to 4) were identifiedfrom human retina by a combination of random cDNA sequencing, data basesearches and homology-based PCR (Smallwood, P. M. et al., Proc. Natl.Acad. Sci. USA 93:9850-9857 (1996)). FGF-15 was identified as adownstream target of a chimeric homeodomain oncoprotein (McWhirter, J.R. et al., Development 124:3221-3232 (1997)). FGF-16, FGF-17, and FGF-18were identified from rat heart and embryos by homology-based PCR,respectively (Miyake, A. et al., Biochem. Biophys. Res. Commun.243:148-152 (1998); Hoshikawa, M. et al., Biochem. Biophys. Res. Commun.244:187-191 (1998); Ohbayashi, N. et al., J. Biol. Chem. 273:18161-18164(1998)). Recently, FGF-19 was identified from human fetal brain by database search (Nishimura, T. et al., Biochim. Biophys. Acta 1444:148-151(1999)). They have a conserved ˜120-amino acid residue core with ˜30 to60% amino acid identity. These FGFs also appear to play important rolesin both developing and adult tissues. Thus, there is a need in the artfor additional FGF molecules having functions and activities that differfrom the known FGFs and for FGF molecules specifically expressed inregions of the brain implicated in human disease.

SUMMARY OF THE INVENTION

The present invention provides a composition comprising an isolatedpolynucleotide selected from the group consisting of:

-   -   (a) a polynucleotide comprising at least eight contiguous        nucleotides of SEQ ID NO:1 or 3;    -   (b) a polynucleotide that encodes a variant of the polypeptide        encoded by (a); and    -   (c) a polynucleotide encoding a protein expressed by a        polynucleotide having the sequence of SEQ ID NO:1 or 3.

The invention further provides for the use of the isolatedpolynucleotides or fragments thereof as diagnostic probes or as primers.

The present invention also provides a composition comprising apolypeptide, wherein said polypeptide is selected from the groupconsisting of:

-   -   (a) a polypeptide comprising at least 6 contiguous amino acids        encoded by SEQ ID NO:1 or 3;    -   (b) a polypeptide encoded by a polynucleotide comprising SEQ ID        NO:1 or 3; and    -   (c) a variant of the polypeptide of (a) or (b).

Polypeptides of the invention are shown in SEQ ID NO:2 and 4. Otherpolypeptides comprise fragments of SEQ ID NO:2 and 4.

In certain preferred embodiments of the invention, the polynucleotide isoperably linked to an expression control sequence. The invention furtherprovides a host cell, including bacterial, yeast, insect and mammaliancells, transformed with the polynucleotide sequence. The invention alsoprovides full-length cDNA and full-length polynucleotides correspondingto SEQ ID NO:1 or 3.

Protein and polypeptide compositions of the invention may furthercomprise a pharmaceutically acceptable carrier. Compositions comprisingan antibody that specifically reacts with such protein or polypeptideare also provided by the present invention.

The invention also provides for the production of large amounts ofotherwise minor cell populations of cells to be used for generation ofcDNA libraries for the isolation of rare molecules expressed in theprecursors cell or progeny; cells produced by treatment may directlyexpress growth factors or other molecules, and conditioned media isscreened in assays for novel activities.

The invention further provides for the isolation, self-renewal andsurvival of mammalian neural stem cells and the differentiation of theirprogeny.

The invention also provides for compositions and methods of preventingor slowing degeneration of or increasing the numbers of dopaminergicneurons, such as in the substantial nigra, in disease states includingParkinson's disease.

The invention further provides for compositions and methods ofpreventing or slowing degeneration of, or for enhancing the growth of,cells in the inner ear.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid sequence comparison of rat FGF-20 with rat FGF-9 andFGF-16. Numbers refer to amino acid positions of FGF-9, FGF-16 andFGF-20. Asterisks indicate identical amino acid residues of thesequences.

FIG. 2. The apparent evolutionary relationships of 20 members of the FGFfamily. The length of each horizontal line is proportional to the degreeof amino acid sequence divergence. mFGF, rFGF, and hFGF indicate mouseFGF, rat FGF, and human FGF, respectively.

FIG. 3. Localization of FGF-20 mRNA in rat brain. (A, B) Coronalsections were hybridized with ³⁵S-labeled FGF-20 antisense (A) and sense(B) probes, and exposed to X-ray film for 10 days. Section A is adjacentto section B. Scale bar=0.5 cm. (C, D) Sections A and B were dipped inliquid emulsion and counterstained with Cresyl violet after 3 weeks.Dark-field photographs of the SNC in sections A and B are shown in C andD, respectively. White grains in the dark-field photograph show thelocalization of FGF-20 mRNA. Scale bar=50 μm. SNC, substantial nigrapars compacta.

FIG. 4. Expression of FGF-20 mRNA in rat brain. Rat brain poly (A)⁺RNA(10 μg) was electrophoresed on a denaturing agarose gel (1%) containingformaldehyde and transferred onto a nitrocellulose membrane.Hybridization was performed with a ³²P labeled rat FGF-20 or β-actincDNA probe. 28S and 18S indicate the positions of 28 and 18S rRNAs,respectively.

FIG. 5. FGF-20 enhances survival of midbrain dopaminergic neurons. (A)Effect of FGF-20 on survival of midbrain dopaminergic neurons inserum-free medium. Midbrain cultured cells were incubated for 4 days inmedium supplemented with 10% horse serum (control) or serum-free mediumsupplemented with FGF-20, and then the numbers of surviving dopaminergicneurons were determined. (B) Effect of FGF-20 for 24 h, and then treatedwith no (control) or 1 mM glutamate for 10 min. The cultured cells werefurther incubated in the medium in the absence of glutamate and FGF-20for 3 days, and then the numbers of surviving dopaminergic neurons weredetermined.

FIG. 6. FIG. 6 provides the DNA sequence (SEQ ID NO:1) of rat FGF-20.

FIG. 7. FIG. 7 provides the amino acid sequence (SEQ ID NO:2) of ratFGF-20.

FIG. 8. FIG. 8 provides the DNA (SEQ ID NO:3) and amino acid (SEQ IDNO:4) sequences of human FGF-20.

FIG. 9. FIG. 9 provides an alignment of the amino acid sequences ofhuman (SEQ ID NO:4) and rat (SEQ ID NO:2) FGF-20.

FIG. 10. FIG. 10 provides codon usage for E. coli.

FIG. 11. FIG. 11 provides codon usage for yeast. The first field ofinformation on each line of the table contains a three-letter code foran amino acid. The second field contains an unambiguous codon for thatamino acid. The third field lists the number of occurrences of thatcodon in the genes from which the table is compiled. The fourth fieldlists the expected number of occurrences of that codon per 1,000 codonsin genes whose codon usage is identical to that compiled in the codonfrequency table. The last field contains the fraction of occurrences ofthe codon in its synonymous codon family.

FIG. 12. FIG. 12 provides codon usage for Drosophila.

DETAILED DESCRIPTION OF THE INVENTION

Because of their potent activities for promoting growth, proliferation,survival and differentiation of a wide variety of cells and tissuetypes, FGFs continue to be pursued as therapeutic agents for a number ofdifferent indications, including wound healing, such as musculo-skeletalconditions, for example, bone fractures, ligament and tissue repair,tendonitis, bursitis, etc.; skin conditions, for example, burns, cuts,lacerations, bed sores, slow healing ulcers, etc.; tissue protection andrepair during myocardial infarction and ischemia, in the treatment ofneurological conditions, for example, neuro-degenerative disease andstroke, in the treatment of eye disease, including macular degeneration,and the like.

The fibroblast growth factor (FGF) proteins identified to date belong toa family of signaling molecules that regulate growth and differentiationof a variety of cell types. The significance of FGF proteins to humanphysiology and pathology relates in part to their key roles inembryogenesis, in blood vessel development and growth, and in bonegrowth. In vitro experiments have demonstrated a role for FGF inregulating cell growth and division of endothelial cells, vascularsmooth muscle cells, fibroblasts, and cardiac and skeletal myocytes.Other members of the FGF family and their biological roles are describedin Crossley et al., Development 121:439-451 (1995); Ohuchi et al.,Development 124:2235-2244 (1997); Gemel et al., Genomics 35:253-257(1996); and Ghosh et al., Cell Growth and Differentiation 7:1425-1434(1996).

FGF proteins are also significant to human health and disease because ofa role in cancer cell growth. For example, FGF-8 was identified as anandrogen-induced growth factor in breast and prostate cancer cells.(Tanaka et al., FEBS Lett. 363:226-230 (1995) and P.N.A.S. 89:8928-8932(1992)).

The role of FGF in normal development is being elucidated in partthrough studies of FGF receptors. Wilke, T. et al., Dev. Dynam.210:41-52 (1997) found that FGFR1, FGFR2, and FGFR3 transcripts werelocalized to specific regions of the head during embryonic developmentin chickens. The expression pattern correlated with areas affected byhuman FGFR mutations in Crouzon syndrome, a condition of abnormalintramembranous bone formation. Belluardo, N. et al., Jour. Comp. Neur.379:226-246 (1997) studied localization of FGFR 1, 2, and 3 mRNAs in ratbrain, and found cellular specificity in several brain regions.Furthermore, FGFR1 and FGFR2 mRNAs were expressed in astroglial reactivecells after brain lesion, supporting a role of certain FGF's in braindisease and injury. Ozawa, K. et al., Mol. Brain Res. 41:279-288 (1996)reported that FGF1 and FGF-5 expression increased after birth, whereasFGF3, FGF-6, FGF-7, and FGF-8 genes showed higher expression in lateembryonic stages than in postnatal stages.

New members of the FGF family are described here, wherein the FGFprotein is expressed in dopaminergic neurons of the substantial nigraand in cochlear tissue of rat embryos. A polynucleotide encoding the ratFGF of the invention has the sequence as shown in SEQ ID NO:1. Apolynucleotide encoding the human FGF of the invention has the sequenceas shown in SEQ ID NO:3. The rat polynucleotide was identified asencoding a member of the FGF family by the conserved regions throughoutthe amino acid sequence and by the regions of homology shared by thepolynucleotides and genes encoding known FGF proteins.

The inventors believe that FGF-20 is a previously unidentified member ofthe FGF family. To date, over 19 human FGF proteins have beenidentified. In most cases, homologous proteins in the other mammals,particularly mice and rats, have also been identified. The humanproteins vary to different degrees in terms of amino acid sequence,receptor specificity, tissue expression patterns, and biologicalactivity.

The present FGF-20 differs in sequence from all the FGF proteinsdescribed to date in publications. FGF-20 shares some homology withFGF-9 and FGF-16.

As discussed herein, the knowledge about the roles played by various FGFproteins continues to grow, but is by far incomplete.

The present invention adds to this knowledge by disclosing that the FGFof SEQ ID NO:1 is highly expressed in dopaminergic neurons of thesubstantia nigra of brain, and human FGF-20 may play a role indevelopment of and recovery from a neural disease, such as Parkinson'sdisease. FGF-20 is also preferentially expressed in rat embryo (E14.5)cochlea of the inner ear.

The invention therefore is based upon the identification, isolation,sequencing and expression patterns of a new fibroblast growth factor(FGF-20).

Isolation and Analysis of Rat cDNA encoding FGF-20 Members of the FGFfamily have a conserved ˜120-amino acid residue core with ˜30 to 70%amino acid identity. Among the members of the FGF family, FGF-9 andFGF-16 are highly homologous (73% amino acid identity). According to theinvention, DNA encoding a novel rat FGF has been identified. Thenucleotide sequence of the entire coding region was determined byadaptor-ligation mediated polymerase chain reaction using rat-brain cDNAas a template and cassette-ligation mediated polymerase chain reactionusing rat genomic DNA as a template. The nucleotide sequence of thecoding region allowed for the elucidation of the complete amino acidsequence of the FGF (212 amino acids), which has a conserved amino acidresidue core (amino acids 62 to 197) (FIG. 1). Two cysteine residuesthat are well conserved in the FGF family are also conserved in theprotein (amino acids 71 and 137) (FIG. 1). This protein is tentativelynamed FGF-20. FGF-20 is most similar to FGF-9 and FGF-16 (70 and 62%amino acid identity) among 19 members of the FGF family, respectively(FIG. 1). The apparent evolutionary relationships of twenty members ofthe FGF family are shown in FIG. 2. FGF-20 was closest to FGF-9 andFGF-16.

Expression of FGF-20 mRNA in Rat Tissues FGF-9 and FGF-16 mRNAs arepreferentially expressed in rat kidney and heart, respectively(Miyamoto, M. et al., Mol. Cell. Biol. 13:4251-4259 (1993); Miyake, A.et al., Biochem. Biophys. Res. Commun. 243:148-152 (1998)). Theexpression of FGF-20 mRNA was examined in adult rat major tissuesincluding brain, heart, lung, liver, kidney, and small intestine bypolymerase chain reaction. FGF-20 mRNA was detected in the brain, butwas undetectable or present in very low levels in other tissues. Toconfirm the expression of FGF-20 mRNA in rat brain, rat brain poly (A)⁺RNA was examined by Northern blotting analysis using a ³²P-labeled ratFGF-20 cDNA probe. A faint but definite signal of FGF-20 mRNA wasdetected (FIG. 4). To confirm the integrity of the poly (A)⁺ RNA, thehybridized probe was washed from the membrane, and the membrane wasrehybridized with a ³²P-labeled rat β-actin cDNA probe. A strong anddiscrete signal of β-actin mRNA was detected indicating that the poly(A)⁺ RNA was not degraded (FIG. 4). FGF-20 mRNA was also detected in ratembryos (E14.5), specifically in the cochlea of the inner ear, using³⁵S-labeled FGF-20 anti-sense and sense cRNA probes (FIG. 10).

Expression of FGF-20 mRNA in Rat Brain To examine the expression ofFGF-20 mRNA in rat brain, consecutive coronal sections of rat brain wereanalyzed by in situ hybridization with an ³⁵S-labeled antisense or senseFGF-20 cRNA probe. Discrete specific labeling was observed only in thesubstantia nigra pars compacta (FIG. 3A, C). No specific labeling wasobserved in other brain regions examined. The cellular localization ofFGF-20 mRNA was examined by microscopy at higher magnification. ByNissle staining of brain sections, glial cells can be identified assmall intensely stained (dark) cells, while neurons are generally largerand less intensely stained (lighter) owing to their larger volume(Gerfen, C. R., Methods in Neurosciences, Academic Press, San Diego,Calif., Vol. 1, pp. 79-97 (1989)). Black grains of labeled probes werefound in most neurons of these brain areas (FIG. 3E). Dopaminergicneurons in the substantial nigra are preferentially localized in thesubstantial nigra pars compacta (Fallon, J. H. et al., The Rat NervousSystem, 2^(nd) Ed., Academic Press, San Diego, Calif., pp. 215-238(1995)). Furthermore, neurons in the substantia nigra pars compactapredominantly consist of dopaminergic neurons (Fallon, J. H. et al., TheRat Nervous System, 2^(nd) Ed., Academic Press, San Diego, Calif., pp.215-238 (1995)). It is expected that FGF-20 is preferentially expressedin dopaminergic neurons in the substantia nigra pars compacta.

Preparation of Recombinant Rat FGF-20 To produce recombinant rat FGF-20,High Five insect cells were infected with recombinant baculoviruscontaining the rat FGF-20 cDNA with the 3′-terminal extension encoding Eand His₆ tags. To detect recombinant FGF-20 in the culture medium, themedium was examined by Western blotting analysis with anti-E tagantibodies. A major band of 26.5 kDa was detected in the culture medium.The observed molecular mass of the major band was consistent with thecalculated molecular mass of recombinant FGF-20 (26,247). This resultindicates that FGF-20 is secreted, although on hydropathy plot analysis(Nielsen, H. et al., Protein Engineering 10:1-6 (1997)) the value of theamino-terminal region of FGF-20 was low, suggesting that FGF-20 has nosignal sequence. Although FGF-9 and FGF-16 have no typical signalsequence in their amino termini, they are also secreted (Miyamoto, M. etal., Mol. Cell. Biol. 13:4251-4259 (1993), Miyake, A. et al., Biochem.Biophys. Res. Commun. 243:148-152 (1998)). Recombinant FGF-20 waspurified from the culture medium by affinity chromatograph with Ni-NTAagarose and was analyzed by SDS-polyacrylamide gel electrophoresis underreducing conditions. A 26.5 kDa protein of FGF-20 was detected.

Neurotrophic Activity of FGF-20 for Rat Midbrain Dopaminergic NeuronsFGFs are local signal molecules that act on proximal cells (Burgess, W.H. et al., Annu. Rev. Biochem. 58:575-606 (1989)). Therefore, it wasexpected that FGF-20 acts on dopaminergic neurons in the substantianigra in autocrine and/or paracrine manner. The neurotrophic activity ofFGF-20 for cultured rat midbrain dopaminergic neurons was examined. Whenthe dopaminergic neurons were cultured in a serum-free medium for 4days, numbers of surviving dopaminergic neurons were greatly reduced.FGF-20 significantly enhanced survival of the dopaminergic neurons inthe serum-free medium (FIG. 5A). The effect of FGF-20 onglutamate-induced neuronal death in cultured rat midbrain dopaminergicneurons was also examined. When the cultured cells were exposed to 1 mMglutamate for 10 min, the numbers of surviving dopaminergic neurons werereduced. FGF-20 also significantly enhanced survival of the dopaminergicneurons exposed to toxic concentrations of glutamate (FIG. 5B).

Several FGFs are expressed in brain and expected to play important rolesas neutrophic factors. FGF-1 and FGF-2 are abundant in brain(Gospodarowicz, D., Methods Enzymol. 147:106-119 (1987)) and exertsurvival enhancing effects on primary cultures from various regions ofthe brain (Walicke, P. A., J. Neurosci. 8:2618-2627 (1988)). FGF-1 isexpressed predominantly in motor and sensory neurons of the midbrain andbrainstem (Elde, R. et al., Neuron 7:349-364 (1991)). In contrast, FGF-2is preferentially expressed in neurons in restricted regions includingthe cingulate cortex, industium grieum, fasciola cinererum andhippocampus, and in astrocytes in widespread regions of the brain(Emoto, N. et al., Growth Factors 2:21-29 (1989); Woodward, W. R. etal., J. Neurosci. 12:142-152 (1992)). FGF-5 is weakly expressed in thecerebral cortex, hippocampus and thalamus (Haub, O. et al., Proc. Natl.Acad. Sci. USA 87:8022-8026 (1990)). FGF-9 and FGF-11 to FGF-14 areexpressed in neurons of restricted regions including the hippocampus,thalamus, midbrain and brainstem (Yamamoto, S. et al., Biochim. Biophys.Acta 1398:38-41 (1998)). In contrast, FGF-20 of the invention waspreferentially expressed in dopaminergic neurons of the substantianigra. The expression profile of FGF-20 was quite distinct from those ofother FGFs, indicating that FGF-20 plays a unique role in the brain.

Degeneration of dopaminergic neurons in the substantia nigra causesParkinson's disease (Fallon, J. H. et al., The Rat Nervous System,2^(nd) Ed., Academic Press, San Diego, Calif., pp. 215-238 (1995)).Therefore, neurotrophic factors for dopaminergic neurons in thesubstantia nigra have received substantial attention. GDNF, Persephin,Artemin, BDNF, and NT-3 enhance survival of midbrain dopaminergicneurons (Lin, L.-F. H. et al., Science 260:1130-1132 (1993); Milbrandt,J. et al., Neuron 20:245-253 (1998); Baloh, R. H. et al., Neuron21:1291-1302 (1998); Hyman, C. et al., Nature 350:230-232 (1991); Hyman,C. et al., J. Neurosci. 14:335-347 (1994)). However, their expression isnot restricted to the substantia nigra (Pochon, N. A. et al., Eur. J.Neurosci. 9:463-471 (1997); Milbrandt, J. et al., Neuron 20:245-253(1998); Baloh, R. H. et al., Neuron 21:1291-1302 (1998); Ernfors, P. etal., Neuron 5:511-526 (1990)). In contrast, the expression of FGF-20,which also enhanced the survival of midbrain dopaminergic neurons, washighly restricted in dopaminergic neurons in the substantia nigra parscompacta. Therefore, FGF-20 is expected to play an important role as aneurotrophic factor for dopaminergic neurons in the substantia nigra. Itis therefore an important finding of the invention that FGF-20 is thefirst neurotrophic factor documented to be expressed preferentially indopaminergic neurons of the substantia nigra.

It is believed that dopamine neurons are dysfunctional for, perhaps,years, before they are irreversibly damaged. (Dunnett, S. B. et al.,Nature 399:A32-A39 (1999)) Thus, neurotrophic agents such as FGF-20 maybe useful in preventing cell death or restoring function The FGF-20 maybe administered using gene transfer methods to block degeneration. Suchmethods have been used with neurotrophic factor GDNF (glial cellline-derived neutrophic factor). In a rat Parkinson's model, nanogramamounts of BDNF and GDNF were measured from transduced cells, and theneuroprotective effect was in the order of 40-70% rescue of nigraldopamine neurons. Thus, transplants using fibroblasts or fibroblast celllines engineered to secrete FGF-20 of the invention can allow secretionof the factor and rescue of nigral dopamine neurons. Alternatively,injection of the striatum or the substantia nigra region with viralvectors carrying the FGF-20 gene may also have a neuroprotective effect.

In Parkinson's Disease, neuronal degeneration in the substantial nigragenerally is slow and protracted. This suggests that early interventioncould block or slow down the degenerative process, perhaps up to 4 or 5years before clinical symptoms appear. A decline in striatal dopaminefunction can be detected by PET and SPECT imaging before the appearanceof clinical symptoms, providing an opportunity for neuroprotectiveintervention at this early stage.

In vivo imaging of dopaminergic activity in the basal ganglia, using[¹⁸F]fluorodopa PET, can be used to monitor progress of the disease aswell as the impact of treatment. A progressive reduction in fluorodopasignal is seen in brain tissue of pre-symptomatic and symptomaticindividuals. After treatment with nigral tissue implant, the fluorodopasignal increases over time. (Dunnett et al., supra.) This and othertechniques known in the art can be used to measure the effect oftreatments described herein using FGF-20, and the clinician will beskilled in the art of determining appropriate treatment levels andregimens.

FGF-20 Expression in Rat Embryo Cochlea FGF-20 is preferentiallyexpressed in the cochlea of the inner ear in rat embryos (E14.5). Thissupports a role for FGF-20 in the development and maintenance of normalear function. Other previously-identified members of the FGF familycontribute to normal ear growth and development. For example, sensorycells in the cochlea of the rat transiently express FGF-1 during thetime of terminal innervation in the sensory epithelium (Dazert et al.,J. Cell Physiol. 177:123-129 (1998)). These authors also found that invitro, spiral ganglion explants cultured in the presence of FGF-1exhibited a dose-dependent increase in the number and length ofneurites. In chick cochlea, FGF-1 mRNA levels increased in sensoryepithelium of the cochlea in response to ototoxic damage, suggestingthat the FGF system may be involved in the response of the cochlearepithelium to ototoxic damage. Pickles et al., Dev. Neuroscience19:476-487 (1997). FGF-2 may help to regulate the proliferation stepduring hair cell development and regeneration after trauma in rats.Zheng et al., J. Neuroscience 17:216-226 (1997). Thus, FGF moleculesplay several roles in maintaining normal development and function of thecochlea, and recovery of the cochlea from ototoxic damage. The absenceof FGF receptor 3 contributes to inner ear defects in mice homozygousfor skeletal and inner ear defects, including failure of pillar celldifferentiation and tunnel of Corti formation, and profound deafness.Colvin et al., Nat. Genet. 12:390-397 (1996). It is of interest thatFGF-20 of the invention binds to FGF receptor 3c (Example 12). The factthat FGF-20 is expressed at a specific stage in rat inner eardevelopment further suggests its importance in development of thistissue.

FGF-20 therefore may be suitable for treating a variety of conditionsrelated to the ear. Currently, about 7.8 million Americans have mildhearing loss, 10 million have moderate hearing loss, and 2.7 millionhave profound or severe hearing loss. The causes include, but are notlimited to, otosclerosis; Cogan's syndrome; Meniere's disease; Pendred'ssyndrome; diabetes-associated hearing loss (non-insulin-dependentdiabetes mellitus in combination with obesity can cause tissue changesin the cochlea, McQueen et al., J. Laryngol. Otol. 113:113-118 (1999));congenital malformations; autoimmune disease-related hearing loss;age-related hearing loss; deafness associated with lack of FGF receptor(Colvin et al., Nat. Genet. 12:390-397 (1996)); ischemia-related hearingdisturbance; and other conditions in which cochlear structure andfunction plays a role. Administration of FGF-20 protein orpolynucleotide may be used to treat inherited, congenital and acquireddiseases of hearing and balance, by promoting the survival,proliferation or differentiation of cells of the inner ear.

Reference to FGF-20 herein is intended to be construed to include growthfactors of any origin which are substantially homologous to and whichare biologically equivalent to the FGF-20 characterized and describedherein. Such substantially homologous growth factors may be native toany tissue or species and, similarly, biological activity can becharacterized in any of a number of biological assay systems.

The term “biologically equivalent” is intended to mean that thecompositions of the present invention are capable of demonstrating someor all of the same growth properties in a similar fashion, notnecessarily to the same degree as the FGF-20 isolated as describedherein or recombinantly produced human FGF-20 of the invention.

By “substantially homologous” it is meant that the degree of homology ofhuman FGF-20 to FGF-20 from any species is greater than that betweenFGF-20 and any previously reported member of the FGF family.

Sequence identity or percent identity is intended to mean the percentageof same residues between two sequences, referenced to human FGF whendetermining percent identity with non-human FGF-20, referenced to FGF-20when determining percent identity with non-FGF-20 growth factors, whenthe two sequences are aligned using the Clustal method (Higgins et al.,Cabios 8:189-191 (1992)) of multiple sequence alignment in the Lasergenebiocomputing software (DNASTAR, INC, Madison, Wis.). In this method,multiple alignments are carried out in a progressive manner, in whichlarger and larger alignment groups are assembled using similarity scorescalculated from a series of pairwise alignments. Optimal sequencealignments are obtained by finding the maximum alignment score, which isthe average of all scores between the separate residues in thealignment, determined from a residue weight table representing theprobability of a given amino acid change occurring in two relatedproteins over a given evolutionary interval. Penalties for opening andlengthening gaps in the alignment contribute to the score. The defaultparameters used with this program are as follows: gap penalty formultiple alignment=10; gap length penalty for multiple alignment=10;k-tuple value in pairwise alignment=1; gap penalty in pairwisealignment=3; window value in pairwise alignment=5; diagonals saved inpairwise alignment=5. The residue weight table used for the alignmentprogram is PAM250 (Dayhoff et al., in Atlas of Protein Sequence andStructure, Dayhoff, Ed., NDRF, Washington, Vol. 5, suppl. 3, p. 345,1978).

Percent conservation is calculated from the above alignment by addingthe percentage of identical residues to the percentage of positions atwhich the two residues represent a conservative substitution (defined ashaving a log odds value of greater than or equal to 0.3 in the PAM250residue weight table). Conservation is referenced to human FGF-20 whendetermining percent conservation with non-human FGF-20, and referencedto FGF-20 when determining percent conservation with non-FGF-20 growthfactors. Conservative amino acid changes satisfying this requirementare: R-K; E-D, Y-F, L-M; V-I, Q-H.

The invention provides FGF-20 proteins or variants thereof having one ormore polymers covalently attached to one or more reactive amino acidside chains. By way of example, not limitation, such polymers includepolyethylene glycol (PEG), which can be attached to one or more freecysteine sulfhydryl residues, thereby blocking the formation ofdisulfide bonds and aggregation when the protein is exposed to oxidizingconditions. In addition, pegylation of FGF-20 proteins and/or muteins isexpected to provide such improved properties as increased half-life,solubility, and protease resistance. FGF-20 proteins and/or muteins mayalternatively be modified by the covalent addition of polymers to freeamino groups such as the lysine epsilon or the N-terminal amino group.Preferred cysteines and lysines for covalent modification will be thosenot involved in receptor or heparin binding. In both human and ratFGF-20, the heparin binding site comprises amino acids 170-186. It willbe apparent to one skilled in the art that the methods for assayingFGF-20 biochemical and/or biological activity may be employed in orderto determine if modification of a particular amino acid residue affectsthe activity of the protein as desired.

It may be advantageous to improve the stability of FGF-20 by modifyingone or more protease cleavage sites. Thus, the present inventionprovides FGF-20 variants in which one or more protease cleavage site hasbeen altered by, for example, substitution of one or more amino acids atthe cleavage site in order to create as FGF-20 variant with improvedstability. Such improved protein stability may be beneficial duringprotein production and/or therapeutic use.

Suitable protease cleavage sites for modification are well known in theart and likely will vary depending on the particular applicationcontemplated. For example, typical substitutions would includereplacement of lysines or arginines with other amino acids such asalanine. The loss of activity, such as receptor binding or heparinbinding, can be tested for as described herein.

FGF-20 can also include hybrid and modified forms of FGF-20 includingfusion proteins and FGF-20 fragments and hybrid and modified forms inwhich certain amino acids have been deleted or replaced andmodifications such as where one or more amino acids have been changed toa modified amino acid or unusual amino acid and modifications such asglycosylations so long as the hybrid or modified form retains thebiological activity of FGF-20. By retaining the biological activity, itis meant that neuronal survival is promoted, although not necessarily atthe same level of potency as that of the FGF-20 isolated as describedherein or that of the recombinantly produced human FGF-20. Fusionproteins can consist of the FGF-20 of the invention or fragment thereofand a signal sequence of a heterologous protein to promote secretion ofthe protein product.

Fusion proteins comprising FGF-20 or a biologically active or antigenicfragment thereof can be produced using methods known in the art. Suchfusion proteins can be used therapeutically or can be produced in orderto simplify the isolation and purification procedures. Histidineresidues can be incorporated to allow immobilized metal affinitychromatography purification. Residues EQKLISEEDL contain the antigenicdeterminant recognized by the myc monoclonal antibody and can beincorporated to allow myc monoclonal antibody-based affinitypurification. A thrombin cleavage site can be incorporated to allowcleavage of the molecule at a chosen site; a preferred thrombin cleavagesite consists of residues LVPRG. Purification of the molecule can befacilitated by incorporating a sequence, such as residues SAWRHPQFGG,which binds to paramagnetic streptavidin beads. Such embodiments aredescribed in WO 97/25345, which is incorporated by reference.

The invention further includes chimeric molecules between FGF-20 andkeratinocyte growth factor (KGF) (Reich-Slotky, R. et al., J. Biol.Chem. 270:29813-29818 (1995)). The chimeric molecule can containspecific regions or fragments of one or both of the FGF-20 and KGFmolecules, such as the FGF-20 fragments described below.

The invention also includes fragments of FGF-20. Preferred fragments ofSEQ ID NO:4 and 2 include: amino acids from about 170 to about 186;amino acids from about 1 to about 169; amino acids 2-211 (212 for SEQ IDNO:2); amino acids from about 1 to about 169 and about 187 to about 211(212 for SEQ ID NO:2), wherein amino acids about 169 and about 187 arejoined by a peptide bond; and amino acids from about 59 to about 193.Such fragments can be prepared from the protein by standard biochemicalmethods or by expressing a polynucleotide encoding the fragment.

FGF-20, or a fragment thereof, can be produced as a fusion proteincomprising human serum albumin (HSA) or a portion thereof. Such fusionconstructs are suitable for enhancing expression of the FGF-20, orfragment thereof, in an eukaryotic host cell. Exemplary HSA portionsinclude the N-terminal polypeptide (amino acids 1-369, 1-419, andintermediate lengths starting with amino acid 1), as disclosed in U.S.Pat. No. 5,766,883, and publication WO 97/24445, incorporated byreference herein. Other chimeric polypeptides can include a HSA proteinwith FGF-20, or fragments thereof, attached to each of the C-terminaland N-terminal ends of the HSA. Such HSA constructs are disclosed inU.S. Pat. No. 5,876,969, incorporated by reference herein.

Also included with the scope of the invention are FGF-20 molecules thatdiffer from native FGF-20 by virtue of changes in biologically activesites. FGF-20 has a putative heparin binding site at amino acid residues170-186. An FGF-20 molecule that does not bind heparin can be preparedby expressing DNA encoding FGF-20, wherein the corresponding codons foramino acid residues 170-186 have been deleted. Conversely, one or moreadditional heparin binding sites can be added to FGF-20 by, for example,expressing DNA encoding FGF-20 wherein the codons corresponding toresidues 170-186 are inserted at the desired position(s) in the readingframe. DNA encoding FGF-20 with altered receptor binding can likewise beproduced. For example, it may be desirable to alter receptor specificityof FGF-20 by substituting the receptor binding regions of a differentFGF for that of FGF-20.

Also included within the meaning of substantially homologous is anyFGF-20 which may be isolated by virtue of cross-reactivity withantibodies to the FGF-20 described herein or whose encoding nucleotidesequences including genomic DNA, mRNA or cDNA may be isolated throughhybridization with the complementary sequence of genomic or subgenomicnucleotide sequences or cDNA of the FGF-20 herein or fragments thereof.It will also be appreciated by one skilled in the art that degenerateDNA sequences can encode human FGF-20 and these are also intended to beincluded within the present invention, as are mammalian allelic variantsof FGF-20.

Growth factors are thought to act at specific receptors. According tothe invention, FGF-20 and as yet unknown members of this family ofgrowth factors act through specific receptors having distinctdistributions as has been shown for other growth factor families. FGF-20binds to FGF receptor 2 and FGF receptor 3, but does not bind to FGFreceptor 1. Thus, its receptor binding profile differs from FGF-2 andFGF-4, which bind to FGF receptor 1.

A preferred hFGF-20 of the present invention has been identified andisolated in purified form as described. Also preferred is hFGF-20prepared by recombinant DNA technology. By “pure form” or “purifiedform” or “substantially purified form” it is meant that an FGF-20composition is substantially free of other proteins which are notFGF-20.

Recombinant human FGF-20 may be made by expressing the DNA sequencesencoding FGF-20 in a suitable transformed host cell. Using methods wellknown in the art, the DNA encoding FGF-20 may be linked to an expressionvector, transformed into a host cell and conditions established that aresuitable for expression of FGF-20 by the transformed cell.

The DNA encoding FGF-20 can be engineered to take advantage of preferredcodon usage of host cells. Codon usage in Pseudomonas aeruginosa isdescribed in, for example, West et al., Nucleic Acids Res. 11:9323-9335(1988). Codon usage in Saccharomyces cerevisiae is described in, forexample, Lloyd et al., Nucleic Acids Res. 20:5289-5295 (1992). Codonpreference in Corynebacteria and a comparison with E. coli preference isprovided in Malubres et al., Gene 134:15-24 (1993). Codon usage inDrosophila melanogaster is described in, for example, Akashi, Genetics136:927-935 (1994). Codon usage in yeast is also shown in FIG. 11, andcodon usage in Drosophila is show in FIG. 12.

Any suitable expression vector may be employed to produce recombinanthuman FGF-20 such as expression vectors for use in insect cells.Baculovirus expression systems can also be employed. A preferable methodis expression in insect cells, such as Tr5 or Sf9 cells, usingbaculovirus vector.

The present invention includes nucleic acid sequences includingsequences that encode human FGF-20. Also included within the scope ofthis invention are sequences that are substantially the same as thenucleic acid sequences encoding FGF-20. Such substantially the samesequences may, for example, be substituted with codons more readilyexpressed in a given host cell such as E. coli according to well knownand standard procedures. Such modified nucleic acid sequences areincluded within the scope of this invention.

Specific nucleic acid sequences can be modified by those skilled in theart and, thus, all nucleic acid sequences that code for the amino acidsequences of FGF-20 can likewise be so modified. The present inventionthus also includes nucleic acid sequence which will hybridize with allsuch nucleic acid sequences or complements of the nucleic acid sequenceswhere appropriate and encode a polypeptide having the neuronal cellsurvival promoting activities disclosed herein. The present inventionalso includes nucleic acid sequences that encode polypeptides that haveneuronal cell survival promoting activity and that are recognized byantibodies that bind to FGF-20. Preferred methods and epitopes forraising antibodies are described in Example 10.

The present invention also encompasses vectors comprising expressionregulatory elements operably linked to any of the nucleic acid sequencesincluded within the scope of the invention. This invention also includeshost cells of any variety that have been transformed with vectorscomprising expression regulatory elements operably linked to any of thenucleic acid sequences included within the scope of the presentinvention.

Methods are also provided herein for producing FGF-20. Preparation canbe by isolation from conditioned medium from a variety of cell types solong as the cell type produces FGF-20. A second and preferred methodinvolves utilization of recombinant methods by isolating or obtaining anucleic acid sequence encoding FGF-20, cloning the sequence along withappropriate regulatory sequences into suitable vectors and cell types,and expressing the sequence to produce FGF-20.

Although FGF-20 has been described on the basis of its ability toenhance the survival of midbrain dopaminergic neurons, this factor mayact on other cell types as well. Thus, it is likely that FGF-20 can acton other neural cells.

It is also likely that FGF-20 will act on non-neuronal cells to promotetheir survival, growth or function. This expectation is based upon theactivity of known growth factors. Members of the FGF family act on manycell types of different function and embryologic origin.

The inventors herein have identified that FGF-20 is expressed in thebrain, but not in other adult tissues, including heart, lung, liver,kidney and small intestine. This suggests a role for FGF-20 in, forexample, Parkinson's disease and other diseases of neural tissue.

The present invention also includes therapeutic or pharmaceuticalcompositions comprising FGF-20 in an effective amount for treatingpatients with neuronal disease including Parkinson's disease, and amethod comprising administering a therapeutically effective amount ofFGF-20. These compositions and methods are useful for treating a numberof diseases. The compositions and methods herein can also be useful toprevent degeneration and/or promote survival in other non-neuronaltissues as well. One skilled in the art can readily use a variety ofassays known in the art to determine whether FGF-20 would be useful inpromoting survival or functioning in a particular cell type, such asneuronal cells.

In certain circumstances, it may be desirable to modulate or decreasethe amount of FGF-20 expressed. Thus, in another aspect of the presentinvention, FGF-20 anti-sense oligonucleotides can be made and a methodutilized for diminishing the level of expression of FGF-20 by a cellcomprising administering one or more FGF-20 anti-sense oligonucleotides.By FGF-20 anti-sense oligonucleotides reference is made tooligonucleotides that have a nucleotide sequence that interacts throughbase pairing with a specific complementary nucleic acid sequenceinvolved in the expression of FGF-20 such that the expression of FGF-20is reduced. Preferably, the specific nucleic acid sequence involved inthe expression of FGF-20 is a genomic DNA molecule or mRNA molecule thatencodes FGF-20. This genomic DNA molecule can comprise regulatoryregions of the FGF-20 gene, or the coding sequence for mature FGF-20protein. The term complementary to a nucleotide sequence in the contextof FGF-20 antisense oligonucleotides and methods therefor meanssufficiently complementary to such a sequence as to allow hybridizationto that sequence in a cell, i.e., under physiological conditions. TheFGF-20 antisense oligonucleotides preferably comprise a sequencecontaining from about 8 to about 100 nucleotides and more preferably theFGF-20 antisense oligonucleotides comprise from about 15 to about 30nucleotides. The FGF-20 antisense oligonucleotides can also contain avariety of modifications that confer resistance to nucleolyticdegradation such as, for example, modified internucleoside linages(Uhlmann and Peyman, Chemical Reviews 90:543-548 1990; Schneider andBanner, Tetrahedron Lett. 31:335 (1990), which are incorporated byreference), modified nucleic acid bases and/or sugars and the like.

The therapeutic or pharmaceutical compositions of the present inventioncan be administered by any suitable route known in the art including forexample intravenous, subcutaneous, intramuscular, transdermal,intrathecal or intracerebral. Administration can be either rapid as byinjection or over a period of time as by slow infusion or administrationof slow release formulation. For treating tissues in the central nervoussystem, administration can be by injection or infusion into thecerebrospinal fluid (CSF). When it is intended that FGF-20 beadministered to cells in the central nervous system, administration canbe with one or more agents capable of promoting penetration of FGF-20across the blood-brain barrier.

FGF-20 can also be linked or conjugated with agents that providedesirable pharmaceutical or pharmacodynamic properties. For example,FGF-20 can be coupled to any substance known in the art to promotepenetration or transport across the blood-brain barrier such as anantibody to the transferrin receptor, and administered by intravenousinjection (see, for example, Friden et al., Science 259:373-377 (1993),which is incorporated by reference). Furthermore, FGF-20 can be stablylinked to a polymer such as polyethylene glycol to obtain desirableproperties of solubility, stability, half-life and otherpharmaceutically advantageous properties. (See, for example, Davis etal., Enzyme Eng. 4:169-73 (1978); Burnham, Am. J. Hosp. Pharm.51:210-218 (1994), which are incorporated by reference.)

The compositions are usually employed in the form of pharmaceuticalpreparations. Such preparations are made in a manner well known in thepharmaceutical art. One preferred preparation utilizes a vehicle ofphysiological saline solution, but it is contemplated that otherpharmaceutically acceptable carriers such as physiologicalconcentrations of other non-toxic salts, five percent aqueous glucosesolution, sterile water or the like may also be used. It may also bedesirable that a suitable buffer be present in the composition. Suchsolutions can, if desired, be lyophilized and stored in a sterileampoule ready for reconstitution by the addition of sterile water forready injection. The primary solvent can be aqueous or alternativelynon-aqueous. FGF-20 can also be incorporated into a solid or semi-solidbiologically compatible matrix which can be implanted into tissuesrequiring treatment.

The carrier can also contain other pharmaceutically-acceptableexcipients for modifying or maintaining the pH, osmolarity, viscosity,clarity, color, sterility, stability, rate of dissolution, or odor ofthe formulation. Similarly, the carrier may contain still otherpharmaceutically-acceptable excipients for modifying or maintainingrelease or absorption or penetration across the blood-brain barrier.Such excipients are those substances usually and customarily employed toformulate dosages for parenteral administration in either unit dosage ormulti-dose form or for direct infusion into the cerebrospinal fluid bycontinuous or periodic infusion.

Dose administration can be repeated depending upon the pharmacokineticparameters of the dosage formulation and the route of administrationused.

It is also contemplated that certain formulations containing FGF-20 areto be administered orally. Such formulations are preferably encapsulatedand formulated with suitable carriers in solid dosage forms. Someexamples of suitable carriers, excipients, and diluents include lactose,dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calciumphosphate, alginates, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose,methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water,mineral oil, and the like. The formulations can additionally includelubricating agents, wetting agents, emulsifying and suspending agents,preserving agents, sweetening agents or flavoring agents. Thecompositions may be formulated so as to provide rapid, sustained, ordelayed release of the active ingredients after administration to thepatient by employing procedures well known in the art. The formulationscan also contain substances that diminish proteolytic degradation andpromote absorption such as, for example, surface active agents.

Depending on the treatment regimen contemplated, it may be desired tocontrol the rate of release of FGF-20 protein or variant thereof toprovide long-term treatment while minimizing the frequency ofadministration. Such treatment regimens may be desired, for example,where the FGF-20 protein is found to be relatively unstable such thatthe localized concentration of active protein is at an efficacious levelfor an insufficient period of time. Thus, for example, for certaindiseases, it may not be desired or practical to perform repeated andfrequent injections. The major advantages of such sustained releasesystems include targeted local delivery of drugs at a constant rate,less drug required to treat the disease state, minimization of possibleside effects, and enhanced efficacy of treatment. Also, these forms ofdelivery systems are capable of protecting drugs that are unstable invivo and that would normally require a frequent dosing interval. Undersuch circumstances, sustained release may be achieved by one of themethods readily available in the art such as the encapsulation of FGF-20conjugated heparin-Sepharose beads to form heparin-alginate microspheresor the preparation of FGF-20 PLG microspheres.

Heparin-alginate microspheres have been successfully employed for thedelivery of Basic Fibroblast Growth Factor to tissue (Lopez et al.,Journal of Pharmacology and Experimental Therapeutics 282(1):385-390(1997)). Similarly, Alginate/heparin-Sepharose microspheres and filmshave been used as drug carriers to control the release of a basicFGF-saponin conjugate in order to control its release in small doses.Addition of heparin to solutions of bFGF prevents losses in activitythat accompany changes in pH or elevation in temperature. See, forexample, Gospodarowicz et al., J. Cell. Physiol. 128:475-484 (1986).

As disclosed herein, FGF-20 has a heparin binding domain at residues170-186. Accordingly, binding of FGF-20 to heparin may be employed inorder to enhance its stability either during in vivo expression oradministration or in vitro during various stages of proteinpurification. Thus, by the present invention, heparin may be added to asolution of FGF-20 and the activity assayed by the methods disclosedherein.

FGF-20 bound heparin-Sepharose beads may be encapsulated into calciumalginate microspheres to permit the controlled release of theheparin-stabilized FGF-20 protein. For example, microspheres may beconstructed by dropping a mixed solution of sodium alginate with FGF-20bound heparin-Sepharose beads into a hardening solution of calciumchloride. Spheres are formed instantaneously as the mixture enters thehardening solution. The size of the microsphere may be adjusted bypassing the FGF-20 bound heparin-Sepharose beads through a cylinder ofreduced cross-sectional area such as through a hypodermic needle.

Encapsulation efficiency may be determined by comparing the amount ofencapsulated growth factor with that initially present in solution. Forexample, the FGF-20 may be stripped from the heparin-Sepharose beadswith a solution of 3 M NaCl and functional activity assays may beperformed.

The specific dose is calculated according to the approximate body weightor body surface area of the patient or the volume of body space to beoccupied. The dose will also be calculated dependent upon the particularroute of administration selected. Further refinement of the calculationsnecessary to determine the appropriate dosage for treatment is routinelymade by those of ordinary skill in the art. Such calculations can bemade without undue experimentation by one skilled in the art in light ofthe activity disclosed herein in assay preparations of target cells.Exact dosages are determined in conjunction with standard dose-responsestudies. It will be understood that the amount of the compositionactually administered will be determined by a practitioner, in the lightof the relevant circumstances including the condition or conditions tobe treated, the choice of composition to be administered, the age,weight, and response of the individual patient, the severity of thepatient's symptoms, and the chosen route of administration.

In one embodiment of this invention, FGF-20 may be therapeuticallyadministered by implanting into patients vectors or cells capable ofproducing a biologically-active form of FGF-20 or a precursor of FGF-20,i.e., a molecule that can be readily converted to a biological-activeform of FGF-20 by the body. In one approach cells that secrete FGF-20may be encapsulated into semipermeable membranes for implantation into apatient. The cells can be cells that normally express FGF-20 or aprecursor thereof or the cells can be transformed to express FGF-20 or aprecursor thereof. It is preferred that the cell be of human origin andthat the FGF-20 be human FGF-20 when the patient is human. However, theformulations and methods herein can be used for veterinary as well ashuman applications and the term “patient” as used herein is intended toinclude human and veterinary patients.

Cells can be grown ex vivo for use in transplantation or engraftmentinto patients (Muench et al., Leuk & Lymph. 16:1-11 (1994), which isincorporated by reference). In another embodiment of the presentinvention, FGF-20 is used to promote the ex vivo expansion of a cellsfor transplantation or engraftment. Current methods have used bioreactorculture systems containing factors such as erythropoietin, colonystimulating factors, stem cell factor, and interleukins to expandhematopoietic progenitor cells for erythrocytes, monocytes, neutrophils,and lymphocytes (Verfaillie, Stem Cells 12:466-476 (1994), which isincorporated by reference). These stem cells can be isolated from themarrow of human donors, from human peripheral blood, or from umbilicalcord blood cells. The expanded blood cells are used to treat patientswho lack these cells as a result of specific disease conditions or as aresult of high dose chemotherapy for treatment of malignancy (George,Stem Cells 12(Suppl 1):249-255 (1994), which is incorporated byreference). In the case of cell transplant after chemotherapy,autologous transplants can be performed by removing bone marrow cellsbefore chemotherapy, expanding the cells ex vivo using methods that alsofunction to purge malignant cells, and transplanting the expanded cellsback into the patient following chemotherapy (for review, see Rummel andVan Zant, J. Hematotherapy 3:213-218 (1994), which is incorporated byreference). Since FGF-20 is expressed in neural cells, it is believedthat FGF-20 can function to prevent or slow the degeneration ofdopaminergic neurons, such as substantia nigra.

In a number of circumstances it would be desirable to determine thelevels of FGF-20 in a patient. The identification of FGF-20 along withthe data herein showing expression of FGF-20 provides the basis for theconclusion that the presence of FGF-20 serves a normal physiologicalfunction related to cell growth and survival. Indeed, other neurotrophicfactors are known to play a role in the function of neuronal andnon-neuronal tissues. (Scully and Otten, Cell Bol. Int. 19:459-469(1995); Otten and Gadient, Int. J. Devl. Neurosciences 13:147-151(1995), which are incorporated by reference.) Endogenously producedFGF-20 may also play a role in certain disease conditions, particularlywhere there is cellular degeneration such as in neurodegenerativeconditions or diseases. Other neurotrophic factors are known to changeduring disease conditions. For example, in multiple sclerosis, levels ofNGF protein in the cerebrospinal fluid are increased during acute phasesof the disease (Bracci-Laudiero et al., Neuroscience Lett. 147:9-12(1992), which is incorporated by reference) and in systemic lupuserythematosus there is a correlation between inflammatory episodes andNGF levels in sera (Bracci-Laudiero et al., NeuroReport 4:563-565(1993), which is incorporated by reference).

Given that FGF-20 is expressed in adult neural cells and in cochlearcells during embryonic development, it is likely that the level ofFGF-20 may be altered in a variety of conditions and that quantificationof FGF-20 levels would provide clinically useful information.Furthermore, in the treatment of degenerative conditions, compositionscontaining FGF-20 can be administered and it would likely be desirableto achieve certain target levels of FGF-20 in sera, in cerebrospinalfluid or in any desired tissue compartment. It would, therefore, beadvantageous to be able to monitor the levels of FGF-20 in a patient.Accordingly, the present invention also provides methods for detectingthe presence of FGF-20 in a sample from a patient.

The term “detection” as used herein in the context of detecting thepresence of FGF-20 in a patient is intended to include the determiningof the amount of FGF-20 or the ability to express an amount of FGF-20 ina patient, the distinguishing of FGF-20 from other growth factors, theestimation of prognosis in terms of probable outcome of a degenerativedisease and prospect for recovery, the monitoring of the FGF-20 levelsover a period of time as a measure of status of the condition, and themonitoring of FGF-20 levels for determining a preferred therapeuticregimen for the patient.

To detect the presence of FGF-20 in a patient, a sample is obtained fromthe patient. The sample can be a tissue biopsy sample or a sample ofblood, plasma, serum, CSF or the like. FGF-20 is expressed in neuraltissues as discussed in Example 8. Samples for detecting FGF-20 can betaken from this tissue. When assessing peripheral levels of FGF-20, itis preferred that the sample be a sample of blood, plasma or serum. Whenassessing the levels of FGF-20 in the central nervous system a preferredsample is a sample obtained from cerebrospinal fluid or neural tissue.

In some instances it is desirable to determine whether the FGF-20 geneis intact in the patient or in a tissue or cell line within the patient.By an intact FGF-20 gene it is meant that there are no alterations inthe gene such as point mutations, deletions, insertions, chromosomalbreakage, chromosomal rearrangements and the like wherein suchalteration might alter production of FGF-20 or alter its biologicalactivity, stability or the like to lead to disease processes orsusceptibility to cellular degenerative conditions. Thus, in oneembodiment of the present invention a method is provided for detectingand characterizing any alterations in the FGF-20 gene. The methodcomprises providing an oligonucleotide that contains the FGF-20 cDNA,genomic DNA or a fragment thereof or a derivative thereof. By aderivative of an oligonucleotide, it is meant that the derivedoligonucleotide is substantially the same as the sequence from which itis derived in that the derived sequence has sufficient sequencecomplementarily to the sequence from which it is derived to hybridize tothe FGF-20 gene. The derived nucleotide sequence is not necessarilyphysically derived from the nucleotide sequence, but may be generated inany manner including for example, chemical synthesis or DNA replicationor reverse transcription or transcription.

Typically, patient genomic DNA is isolated from a cell sample from thepatient and digested with one or more restriction endonucleases such as,for example, TaqI and AluI. Using the Southern blot protocol, which iswell known in the art, this assay determines whether a patient or aparticular tissue in a patient has an intact FGF-20 gene or an FGF-20gene abnormality.

Hybridization to an FGF-20 gene would involve denaturing the chromosomalDNA to obtain a single-stranded DNA; contacting the single-stranded DNAwith a gene probe associated with the FGF-20 gene sequence; andidentifying the hybridized DNA-probe to detect chromosomal DNAcontaining at least a portion of a human FGF-20 gene.

The term “probe” as used herein refers to a structure comprised of apolynucleotide that forms a hybrid structure with a target sequence, dueto complementarity of probe sequence with a sequence in the targetregion. Oligomers suitable for use as probes may contain a minimum ofabout 8-12 contiguous nucleotides which are complementary to thetargeted sequence and preferably a minimum of about 20.

The FGF-20 gene probes of the present invention can be DNA or RNAoligonucleotides and can be made by any method known in the art such as,for example, excision, transcription or chemical synthesis. Probes maybe labeled with any detectable label known in the art such as, forexample, radioactive or fluorescent labels or enzymatic marker. Labelingof the probe can be accomplished by any method known in the art such asby PCR, random priming, end labeling, nick translation or the like. Oneskilled in the art will also recognize that other methods not employinga labeled probe can be used to determine the hybridization. Examples ofmethods that can be used for detecting hybridization include Southernblotting, fluorescence in situ hybridization, and single-strandconformation polymorphism with PCR amplification.

Hybridization is typically carried out at 25°-45° C., more preferably at32°-40° C. and more preferably at 37°-38° C. The time required forhybridization is from about 0.25 to about 96 hours, more preferably fromabout one to about 72 hours, and most preferably from about 4 to about24 hours.

FGF-20 gene abnormalities can also be detected by using the PCR methodand primers that flank or lie within the FGF-20 gene. The PCR method iswell known in the art. Briefly, this method is performed using twooligonucleotide primers which are capable of hybridizing to the nucleicacid sequences flanking a target sequence that lies within an FGF-20gene and amplifying the target sequence. The terms “oligonucleotideprimer” as used herein refers to a short strand of DNA or RNA ranging inlength from about 8 to about 30 bases. The upstream and downstreamprimers are typically from about 20 to about 30 base pairs in length andhybridize to the flanking regions for replication of the nucleotidesequence. The polymerization is catalyzed by a DNA-polymerase in thepresence of deoxynucleotide triphosphates or nucleotide analogs toproduce double-stranded DNA molecules. The double strands are thenseparated by any denaturing method including physical, chemical orenzymatic. Commonly, a method of physical denaturation is used involvingheating the nucleic acid, typically to temperatures from about 80° C. to105° C. for times ranging from about 1 to about 10 minutes. The processis repeated for the desired number of cycles.

The primers are selected to be substantially complementary to the strandof DNA being amplified. Therefore, the primers need not reflect theexact sequence of the template, but must be sufficiently complementaryto selectively hybridize with the strand being amplified.

After PCR amplification, the DNA sequence comprising FGF-20 or pre-proFGF-20 or a fragment thereof is then directly sequenced and analyzed bycomparison of the sequence with the sequences disclosed herein toidentify alterations which might change activity or expression levels orthe like.

In another embodiment, a method for detecting FGF-20 is provided basedupon an analysis of tissue expressing the FGF-20 gene, as described inthe Examples. The method comprises hybridizing a polynucleotide to mRNAfrom a sample of tissue that normally expresses the FGF-20 gene. Thesample is obtained from a patient suspected of having an abnormality inthe FGF-20 gene or in the FGF-20 gene of particular cells.

To detect the presence of mRNA encoding FGF-20 protein, a sample isobtained from a patient. The sample can be from blood or from a tissuebiopsy sample. The sample may be treated to extract the nucleic acidscontained therein. The resulting nucleic acid from the sample issubjected to gel electrophoresis or other size separation techniques.

The mRNA of the sample is contacted with a DNA sequence serving as aprobe to form hybrid duplexes. The use of a labeled probes as discussedabove allows detection of the resulting duplex.

When using the cDNA encoding FGF-20 protein or a derivative of the cDNAas a probe, high stringency conditions can be used in order to preventfalse positives, that is the hybridization and apparent detection ofFGF-20 nucleotide sequences when in fact an intact and functioningFGF-20 gene is not present. When using sequences derived from the FGF-20cDNA, less stringent conditions could be used, however, this would be aless preferred approach because of the likelihood of false positives.The stringency of hybridization is determined by a number of factorsduring hybridization and during the washing procedure, includingtemperature, ionic strength, length of time and concentration offormamide.

In order to increase the sensitivity of the detection in a sample ofmRNA encoding the FGF-20 protein, the technique of reversetranscription/polymerization chain reaction (RT/PCR) can be used toamplify cDNA transcribed from mRNA encoding the FGF-20 protein. Themethod of RT/PCR is well known in the art, and can be performed asfollows. Total cellular RNA is isolated by, for example, the standardguanidium isothiocyanate method and the total RNA is reversetranscribed. The reverse transcription method involves synthesis of DNAon a template of RNA using a reverse transcriptase enzyme and a 3′ endprimer. Typically, the primer contains an oligo(dT) sequence. The cDNAthus produced is then amplified using the PCR method and FGF-20 specificprimers. (Belyavsky et al., Nucl. Acid Res. 17:2919-2932 (1989); Krugand Berger, Methods in Enzymology 152:316-325, Academic Press, NY, 1987,which are incorporated by reference).

The polymerase chain reaction method is performed as described aboveusing two oligonucleotide primers that are substantially complementaryto the two flanking regions of the DNA segment to be amplified.

Following amplification, the PCR product is then electrophoresed anddetected by ethidium bromide staining or by phosphoimaging.

The present invention further provides for methods to detect thepresence of FGF-20 protein in a sample obtained from a patient. Anymethod known in the art for detecting proteins can be used. Such methodsinclude, but are not limited to immunodiffusion, immunoelectrophoresis,immunochemical methods, binder-ligand assays, immunohistochemicaltechniques, agglutination and complement assays. (For example, see Basicand Clinical Immunology, 217-262, Sites and Terr, eds., Appleton &Lange, Norwalk, Conn., 1991 which is incorporated by reference).Preferred are binder-ligand immunoassay methods including reactingantibodies with an epitope or epitopes of the FGF-20 protein andcompetitively displacing a labeled FGF-20 protein or derivative thereof.Preferred antibodies are prepared according to Example 11.

As used herein, a derivative of the FGF-20 protein is intended toinclude a polypeptide in which certain amino acids have been deleted,replaced, or changed to modified or unusual amino acids wherein theFGF-20 derivative is biologically equivalent to FGF-20 and wherein thepolypeptide derivative cross-reacts with antibodies raised against theFGF-20 protein. By cross-reaction it is meant that an antibody reactswith an antigen other than the one that induced its formation.

Numerous competitive and non-competitive protein binding immunoassaysare well known in the art. Antibodies employed in such assays may beunlabeled, for example as used in agglutination tests, or labeled foruse in a wide variety of assay methods. Labels that can be used includeradionuclides, enzymes, fluorescers, chemiluminescers, enzyme substratesor co-factors, enzyme inhibitors, particles, dyes and the like for usein radioimmunoassay (RIA), enzyme immunoassays, e.g., enzyme-linkedimmunosorbent assay (ELISA), fluorescent immunoassays and the like.

Polyclonal or monoclonal antibodies to the FGF-20 protein or an epitopethereof can be made for use in immunoassays by any of a number ofmethods known in the art. By epitope reference is made to an antigenicdeterminant of a polypeptide. An epitope could comprise 3 amino acids ina spatial conformation which is unique to the epitope. Generally anepitope consists of at least 5 such amino acids. Methods of determiningthe spatial conformation of amino acids are known in the art, andinclude, for example, x-ray crystallography and 2 dimensional nuclearmagnetic resonance.

One approach for preparing antibodies to a protein is the selection andpreparation of an amino acid sequence of all or part of the protein,chemically synthesizing the sequence and injecting it into anappropriate animal, usually a rabbit or a mouse (see Example 11).

Oligopeptides can be selected as candidates for the production of anantibody to the FGF-20 protein based upon the oligopeptides lying inhydrophilic regions, which are thus likely to be exposed in the matureprotein. Oligopeptides for raising antibodies include the contiguousamino acids at positions 176-189, or 56-70 of SEQ ID NO:4. Theseoligopeptides are RDGARSKRHQKFTH (SEQ ID NO:5) and QLAHLHGILRRRQLY (SEQID NO:6). Additional oligopeptides can be determined using, for example,the Antigenicity Index of Welling, G. W. et al., FEBS Lett. 188:215-218(1985), incorporated herein by reference.

Antibodies to FGF-20 can also be raised against oligopeptides thatinclude one or more of the conserved regions identified herein such thatthe antibody can cross-react with other family members. Such antibodiescan be used to identify and isolate the other family members.

Methods for preparation of the FGF-20 protein or an epitope thereofinclude, but are not limited to chemical synthesis, recombinant DNAtechniques or isolation from biological samples. Chemical synthesis of apeptide can be performed, for example, by the classical Merrifeld methodof solid phase peptide synthesis (Merrifeld, J., Am. Chem. Soc. 85:2149(1963), which is incorporated by reference) or the FMOC strategy on aRapid Automated Multiple Peptide Synthesis system (E. I. du Pont deNemours Company, Wilmington, Del.) (Caprino and Han, J. Org. Chem.37:3404 (1972), which is incorporated by reference).

Polyclonal antibodies can be prepared by immunizing rabbits or otheranimals by injecting antigen followed by subsequent boosts atappropriate intervals. The animals are bled and sera assayed againstpurified FGF-20 protein usually by ELISA or by bioassay based upon theability to block the action of FGF-20 on neurons or other cells. Whenusing avian species, e.g., chicken, turkey and the like, the antibodycan be isolated from the yolk of the egg. Monoclonal antibodies can beprepared after the method of Milstein and Kohler by fusing splenocytesfrom immunized mice with continuously replicating tumor cells such asmyeloma or lymphoma cells. (Milstein and Kohler, Nature 256:495-497(1975); Gulfre and Milstein, Methods in Enzymology: ImmunochemicalTechniques 73:1-46, Langone and Banatis eds., Academic Press, 1981 whichare incorporated by reference). The hybridoma cells so formed are thencloned by limiting dilution methods and supernates assayed for antibodyproduction by ELISA, RIA or bioassay.

The unique ability of antibodies to recognize and specifically bind totarget proteins provides an approach for treating an overexpression ofthe protein. Thus, another aspect of the present invention provides fora method for preventing or treating diseases involving overexpression ofthe FGF-20 protein by treatment of a patient with specific antibodies tothe FGF-20 protein.

Specific antibodies, either polyclonal or monoclonal, to the FGF-20protein can be produced by any suitable method known in the art asdiscussed above. For example, murine or human monoclonal antibodies canbe produced by hybridoma technology or, alternatively, the FGF-20protein, or an immunologically active fragment thereof, or ananti-idiotypic antibody, or fragment thereof can be administered to ananimal to elicit the production of antibodies capable of recognizing andbinding to the FGF-20 protein. Such antibodies can be from any class ofantibodies including, but not limited to IgG, IgA, IgM, IgD, and IgE orin the case of avian species, IgY and from any subclass of antibodies.

Polypeptides encoded by the instant polynucleotides and correspondingfull-length genes can be used to screen peptide libraries, proteinlibraries, small molecule libraries, and phage display libraries, andother known methods, to identify analogs or antagonists.

Native FGF polypeptides may play a role in cancer. For example, FGFfamily members can induce marked morphological transformation of NIH 3T3cells, and exhibit strong tumorigenicity in nude mice. Angiogenicactivity has been exhibited by FGF family members. Thus, inhibitors ofFGF can be used to treat cancer.

A library of peptides may be synthesized following the methods disclosedin U.S. Pat. No. 5,010,175, and in PCT No. WO 91/17823. As describedbelow in brief, a mixture of peptides is prepared, which is thenscreened to identify the peptides exhibiting the desired signaltransduction and receptor binding activity. According to the method ofthe '175 patent, a suitable peptide synthesis support (e.g., a resin) iscoupled to a mixture of appropriately protected, activated amino acids.The concentration of each amino acid in the reaction mixture is balancedor adjusted in inverse proportion to its coupling reaction rate so thatthe product is an equimolar mixture of amino acids coupled to thestarting resin. The bound amino acids are then deprotected, and reactedwith another balanced amino acid mixture to form an equimolar mixture ofall possible dipeptides. This process is repeated until a mixture ofpeptides of the desired length (e.g., hexamers) is formed. Note that oneneed not include all amino acids in each step: one may include only oneor two amino acids in some steps (e.g., where it is known that aparticular amino acid is essential in a given position), thus reducingthe complexity of the mixture. After the synthesis of the peptidelibrary is completed, the mixture of peptides is screened for binding tothe selected polypeptide. The peptides are then tested for their abilityto inhibit or enhance activity. Peptides exhibiting the desired activityare then isolated and sequenced.

The method described in PCT No. WO 91/17823 is similar. However, insteadof reacting the synthesis resin with a mixture of activated amino acids,the resin is divided into twenty equal portions (or into a number ofportions corresponding to the number of different amino acids to beadded in that step), and each amino acid is coupled individually to itsportion of resin. The resin portions are then combined, mixed, and againdivided into a number of equal portions for reaction with the secondamino acid. In this manner, each reaction may be easily driven tocompletion. Additionally, one may maintain separate “subpools” bytreating portions in parallel, rather than combining all resins at eachstep. This simplifies the process of determining which peptides areresponsible for any observed receptor binding or signal transductionactivity.

In such cases, the subpools containing, e.g., 1-2,000 candidates eachare exposed to one or more polypeptides of the invention. Each subpoolthat produces a positive result is then resynthesized as a group ofsmaller subpools (sub-subpools) containing, e.g., 20-100 candidates, andreassayed. Positive sub-subpools may be resynthesized as individualcompounds, and assayed finally to determine the peptides that exhibit ahigh binding constant. These peptides can be tested for their ability toinhibit or enhance the native activity. The methods described in PCT No.WO 91/7823 and U.S. Pat. No. 5,194,392 (herein incorporated byreference) enable the preparation of such pools and subpools byautomated techniques in parallel, such that all synthesis andresynthesis may be performed in a matter of days.

Peptide agonists or antagonists are screened using any available method,such as signal transduction, antibody binding, receptor binding andmitogenic assays. The assay conditions ideally should resemble theconditions under which the native activity is exhibited in vivo, thatis, under physiologic pH, temperature, and ionic strength. Suitableagonists or antagonists will exhibit strong inhibition or enhancement ofthe native activity at concentrations that do not cause toxic sideeffects in the subject. Agonists or antagonists that compete for bindingto the native polypeptide may require concentrations equal to or greaterthan the native concentration, while inhibitors capable of bindingirreversibly to the polypeptide may be added in concentrations on theorder of the native concentration.

The availability of hFGF-20 and rFGF-20 allows for the identification ofsmall molecules and low molecular weight compounds that inhibit thebinding of FGF-20 to its receptor, through routine application ofhigh-throughput screening methods (HTS). HTS methods generally refer totechnologies that permit the rapid assaying of lead compounds fortherapeutic potential. HTS techniques employ robotic handling of testmaterials, detection of positive signals, and interpretation of data.Lead compounds may be identified via the incorporation of radioactivityor through optical assays that rely on absorbance, fluorescence orluminescence as read-outs. Gonzalez, J. E., et al., Curr. Opin. Biotech.9:624-631 (1998). Assays for detecting interaction between an FGFmolecule and FGF receptor are described in, for example, Blunt, A. G. etal., J. Biol. Chem. 272:3733-3738 (1997), and such assays can be adaptedfor determining if a candidate molecule can inhibit the interactionbetween FGF-20 and its receptor.

Model systems are available that can be adapted for use in highthroughput screening for compounds that inhibit the interaction ofFGF-20 with receptors to which it binds (see Example 12), for example bycompeting with FGF-20 for receptor binding. Sarubbi et al., Anal.Biochem. 237:70-75 (1996), describe cell-free, non-isotopic assays fordiscovering molecules that compete with natural ligands for binding tothe active site of IL-1 receptor. Martens, C. et al., Anal. Biochem.273:20-31 (1999), describe a generic particle-based nonradioactivemethod in which a labeled ligand binds to its receptor immobilized on aparticle; label on the particle decreases in the presence of a moleculethat competes with the labeled ligand for receptor binding.

The therapeutic FGF-20 polynucleotides and polypeptides of the presentinvention may be utilized in gene delivery vehicles. The gene deliveryvehicle may be of viral or non-viral origin (see generally, Jolly,Cancer Gene Therapy 1:51-64 (1994); Kimura, Human Gene Therapy 5:845-852(1994); Connelly, Human Gene Therapy 1:185-193 (1995); and Kaplitt,Nature Genetics 6:148-153 (1994)). Gene therapy vehicles for delivery ofconstructs including a coding sequence of a therapeutic of the inventioncan be administered either locally or systemically. These constructs canutilize viral or non-viral vector approaches. Expression of such codingsequences can be induced using endogenous mammalian or heterologouspromoters. Expression of the coding sequence can be either constitutiveor regulated.

The present invention can employ recombinant retroviruses which areconstructed to carry or express a selected nucleic acid molecule ofinterest. Retrovirus vectors that can be employed include thosedescribed in EP 0 415 731; WO 90/07936; WO 94/03622; WO 93/25698; WO93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218; Vile andHart, Cancer Res. 53:3860-3864 (1993); Vile and Hart, Cancer Res.53:962-967 (1993); Ram et al., Cancer Res. 53:83-88 (1993); Takamiya etal., J. Neurosci. Res. 33:493-503 (1992); Baba et al., J. Neurosurg.79:729-735 (1993); U.S. Pat. No. 4,777,127; GB Patent No. 2,200,651; andEP 0 345 242. Preferred recombinant retroviruses include those describedin WO 91/02805.

Packaging cell lines suitable for use with the above-describedretroviral vector constructs may be readily prepared (see PCTpublications WO 95/30763 and WO 92/05266), and used to create producercell lines (also termed vector cell lines) for the production ofrecombinant vector particles. Within particularly preferred embodimentsof the invention, packaging cell lines are made from human (such asHT1080 cells) or mink parent cell lines, thereby allowing production ofrecombinant retroviruses that can survive inactivation in human serum.

The present invention also employs alphavirus-based vectors that canfunction as gene delivery vehicles. Such vectors can be constructed froma wide variety of alphaviruses, including, for example, Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532).Representative examples of such vector systems include those describedin U.S. Pat. Nos. 5,091,309; 5,217,879; and 5,185,440; and PCTPublication Nos. WO 92/10578; WO 94/21792; WO 95/27069; WO 95/27044; andWO 95/07994.

Gene delivery vehicles of the present invention can also employparvovirus such as adeno-associated virus (AAV) vectors. Representativeexamples include the AAV vectors disclosed by Srivastava in WO 93/09239,Samulski et al., J. Vir. 63:3822-3828 (1989); Mendelson et al., Virol.166:154-165 (1988); and Flotte et al., P.N.A.S. 90:10613-10617 (1993).

AAV vectors may be suitable for administering FGF-20 to treat hearingdisorders. For example, Lalwani et al., Gene Ther. 3:588-592 (1996),used AAV to obtain in vivo expression of a foreign gene in the cochleaof guinea pigs.

Representative examples of adenoviral vectors include those described byBerkner, Biotechniques 6:616-627 (Biotechniques); Rosenfeld et al.,Science 252:431-434 (1991); WO 93/19191; Kolls et al., P.N.A.S.:215-219(1994); Kass-Eisleret al., P.N.A.S. 90:11498-11502 (1993); Guzman etal., Circulation 88:2838-2848 (1993); Guzman et al., Cir. Res.73:1202-1207 (1993); Zabner et al., Cell 75:207-216 (1993); Li et al.,Hum. Gene Ther. 4:403-409 (1993); Cailaud et al., Eur. J. Neurosci.5:1287-1291 (1993); Vincent et al., Nat. Genet. 5:130-134 (1993); Jaffeet al., Nat. Genet. 1:372-378 (1992); and Levrero et al., Gene101:195-202 (1992). Exemplary adenoviral gene therapy vectors employablein this invention also include those described in WO 94/12649, WO93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655.Administration of DNA linked to killed adenovirus as described inCuriel, Hum. Gene Ther. 3:147-154 (1992), may be employed.

Other gene delivery vehicles and methods may be employed, includingpolycationic condensed DNA linked or unlinked to killed adenovirusalone, for example Curiel, Hum. Gene Ther. 3:147-154 (1992);ligand-linked DNA, for example see Wu, J. Biol. Chem. 264:16985-16987(1989); eukaryotic cell delivery vehicles cells, for example see U.S.Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796;deposition of photopolymerized hydrogel materials; hand-held genetransfer particle gun, as described in U.S. Pat. No. 5,149,655; ionizingradiation as described in U.S. Pat. No. 5,206,152 and in WO 92/11033;nucleic charge neutralization or fusion with cell membranes. Additionalapproaches are described in Philip, Mol. Cell Biol. 14:2411-2418 (1994),and in Woffendin, Proc. Natl. Acad. Sci. 91:1581-1585 (1994).

Naked DNA may also be employed. Exemplary naked DNA introduction methodsare described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptakeefficiency may be improved using biodegradable latex beads. DNA coatedlatex beads are efficiently transported into cells after endocytosisinitiation by the beads. The method may be improved further by treatmentof the beads to increase hydrophobicity and thereby facilitatedisruption of the endosome and release of the DNA into the cytoplasm.Liposomes that can act as gene delivery vehicles are described in U.S.Pat. No. 5,422,120, PCT Patent Publication Nos. WO 95/13796, WO94/23697, and WO 91/14445, and EP No. 0 524 968.

Further non-viral delivery suitable for use includes mechanical deliverysystems such as the approach described in Woffendin et al., Proc. Natl.Acad. Sci. USA 91(24): 11581-11585 (1994). Moreover, the coding sequenceand the product of expression of such can be delivered throughdeposition of photopolymerized hydrogel materials. Other conventionalmethods for gene delivery that can be used for delivery of the codingsequence include, for example, use of hand-held gene transfer particlegun, as described in U.S. Pat. No. 5,149,655; use of ionizing radiationfor activating transferred gene, as described in U.S. Pat. No. 5,206,152and PCT Patent Publication No. WO 92/11033.

FGF has been implicated in diseases characterized by loss of function,inadequate function/number, abnormal function or death of cells, tissuesor organs for which function or survival can be prolonged/rescued, andabnormalities reversed or prevented by therapy with FGF. Loss ofpulmonary, bronchia or alveolar cells or function, healing of pulmonaryor bronchia wounds, pulmonary infraction, emphysema/chronic obstructivepulmonary disease, asthma, sequelae of infectious or autoimmune disease,sequelae of pulmonary arterial or venous hypertension, pulmonaryfibrosis, pulmonary disease of immaturity, and cystic fibrosis areconditions amenable to treatment with FGF. Ischemic vascular disease maybe amenable to FGF-20 treatment, wherein the disease is characterized byinadequate blood flow to an organ(s). Treatment may induce therapeuticangiogenesis or preserve function/survival of cells (myocardialischemia/infarction, peripheral vascular disease, renal artery disease,stroke).

Cardiomyopathies characterized by loss of function or death of cardiacmyocytes or supporting cells in the heart (congestive heart failure,myocarditis) may also be treated using FGF-20, as can musculoskeletaldisease characterized by loss of function, inadequate function or deathof skeletal muscle cells, bone cells or supporting cells. Examplesinclude skeletal myopathies, bone disease, and arthritis.

FGF-20 polynucleotides and polypeptides may aid in correction ofcongenital defects due to loss of FGF-20 molecule or its function(heart, lung, brain, limbs, kidney, etc.). FGF-20 polynucleotides andpolypeptides may also aid in the correction of such defects wherein thedefects lead to hearing loss due to cochlear defects.

Treatment of wound healing is yet another use of FGF-20 polypeptides andpolynucleotides, either due to trauma, disease, medical or surgicaltreatment, including regeneration of cell populations and tissuesdepleted by these processes. Examples include liver regeneration,operative wound healing, re-endothelialization of injured blood vessels,healing of traumatic wounds, healing of ulcers due to vascular,metabolic disease, etc., bone fractures, loss of cells due toinflammatory disease, etc.

FGF-20 may also be used in screens to identify drugs for treatment ofcancers which involve over activity of the molecule, or new targetswhich would be useful in the identification of new drugs.

For all of the preceding embodiments, the clinician will determine,based on the specific condition, whether FGF-20 polypeptides orpolynucleotides, antibodies to FGF-20, or small molecules such aspeptide analogues or antagonists, will be the most suitable form oftreatment. These forms are all within the scope of the invention.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLES Example 1

Preparation of RNA—RNA was prepared from adult rat brain using an RNAextraction kit (Pharmacia Biotech, Uppsala, Sweden). Poly (A)⁺ RNA wasprepared using oligo (dT)-cellulose (Type 2, Collaborative BiomedicalProducts, Bedford, Mass.).

Example 2

Isolation and Analysis of Rat FGF-20 cDNA—DNA was amplified from ratgenomic DNA by polymerase chain reaction (PCR) for 30 cycles in 25 μl ofa reaction mixture containing 5 pmole/μl of each of the sense andantisense degenerate primers representing all possible codonscorresponding to the consensus amino acid sequences of rat FGF-9 (17)and FGF-16 (21), FEENWY and THFLPR, respectively. The amplified productwas further amplified by PCR with each of the sense and antisensedegenerate primers representing all possible codons corresponding toanother consensus amino acid sequences of rat FGF-9 (17) and FGF-16(21), ENWYNT and HQKFTH, respectively. The amplified DNA of expectedsize (approximately 150 base pairs) was cloned into the pGEM-T DNAvector (Promega, Madison, Wis.). The nucleotide sequence of the clonedDNA was determined by a DNA sequencer (Applied Biosystems, Foster,Calif.). To determine the coding region of a novel FGF cDNA, the codingregion was amplified from cDNA synthesized from rat brain poly (A)⁺ RNAby adaptor-ligation mediated polymerase chain reaction using a MarathoncDNA amplification kit (Clontech, Palo Alto, Calif.). To determine theamino-terminal region, DNA encoding the region was amplified from ratgenomic DNA by cassette-ligation mediated polymerase chain reaction(Isegawa, Y. et al., Mol. Cell. Probes 6:467-475 (1992)) using a LA PCRin vitro cloning kit (TaKaRa, Kyoto, Japan). The cDNA encoding theentire coding region of the FGF was amplified from rat brain cDNA bypolymerase chain reaction in the presence of 5% dimethyl sulfoxide(Villarreal, X. C. et al., Anal. Biochem. 197:362-367 (1991)) using theFGF-specific primers including the 5′ and 3′ noncoding sequences, andcloned into the pGEM-T DNA vector. The apparent evolutionaryrelationships of members. of the FGF family were examined by theunweighted pair-group method with arithmetic mean method using thesequence analysis software, Genetyx (Software Development Co., Tokyo,Japan).

Example 3

Expression of FGF-20 mRNA in Rat Embryonic Inner Ear—Expression ofFGF-20 in rat embryos was examined. Consecutive transverse sections ofrat embryos (E14.5) were examined by in situ hybridization with³⁵S-labeled FGF-20 anti-sense and sense cRNA probes. The sections werecounterstained with hematoxylin and eosin. Bright-field and dark-fieldphotographs of the sections reveal that FGF-20 is preferentiallyexpressed in the cochlea of the inner ear.

Example 4

Northern Blotting Analysis—Poly (A)⁺ RNA (10 μg) from rat adult brainwas dissolved on a denaturing agarose gel (1%) containing formaldehyde,and transferred to a nitrocellulose membrane in 20×SSC (1×SSC:0.15 MNACI/0.015 M sodium citrate) overnight. A ³²P-labeled FGF-20 cDNA probe(˜650 base pairs) was labeled with a random primer labeling kit(Pharmacia Biotech, Uppsala, Sweden) and deoxycytidine 5′-[α-³²P-]triphosphate (˜110 TBq/mmol) (ICN Biomedicals Inc., Costa Mesa, Calif.).The membrane was incubated in hybridization solution containing thelabeled probe as described (22), and analyzed with a radio-imaginganalyzer (BAS 2000, Fuji Photo Film Co., Tokyo, Japan). To confirm theintegrity of the poly (A)⁺ RNA, the hybridized probe on the membrane waswashed with 0.5×SSC containing 0.01 M EDTA (pH 8.0) at 100° C. for 5 minand with 0.05×SSC containing 0.01 M EDTA (pH 8.0) and 0.1% SDS at 60° C.for 15 min. The washed membrane was rehybridized with a ³²P-labeled ratPactin cDNA probe (˜410 base pairs) (Nudel, U. et al., Nucleic AcidsRes. 11:1759-1771 (1983)).

Example 5

In Situ Hybridization—Adult Wistar rat brain was frozen in powdered dryice, and sagittal sections were cut at 16 μm with a cryostat,thaw-mounted onto poly-L-lysine-coated slides, and stored at −85° C.until hybridization. A ³⁵S-labeled rat FGF-20 antisense or sense cRNAprobe was transcribed using SP6 RNA polymerase or T7 RNA polymerase(TaKaRa, Kyoto, Japan) with uridine 5′-α[³⁵S]thiotriphosphate (˜30TBq/mmol) (Amersham, Buckinghamshire, England), respectively. Thesections were examined by in situ hybridization with the labeled probeas described (Yamasaki, M. et al., J. Biol. Chem. 271:15918-15921(1996)).

Example 6

Preparation of Recombinant Rat FGF-20—The rat FGF-20 cDNA with a DNAfragment (75 BP) encoding an E-tag (GAPVPYPDPLEPR) and a His₆ tag(HHHHHH) at the 3′-terminus of the coding region was constructed in atransfer vector DNA, pBacPAK9 (Clontech, Palo Alto, Calif.). Recombinantbaculovirus containing the FGF-20 cDNA with the tag sequences wasobtained by cotransfection of Sf9 cells with the recombinant pBacPAK9and a Bsu36 I-digested expression vector, BacPAK6 (Clontech, Palo Alto,Calif.). High Five insect cells were infected with the resultantrecombinant baculovirus and incubated at 27° C. for 65 h in serum-freemedium EX-CELL 400 (JRH Biosciences, Lenexa, Kans.). The culture mediumwas dialyzed against phosphate-buffered saline (PBS), and applied to acolumn of Ni-NTA agarose (QIAGEN GmbH, Hilden, Germany) in PBScontaining 20 mM imidazole and 0.5 M NaCl. After washing the column withPBS containing 20 mM imidazole and 0.5 M NaCl, recombinant FGF-20 waseluted from the column with PBS containing 250 mM imidazole and 0.5 MNaCl, and applied to a column of Bio-Gel P-6 DG (Bio-Rad Lab., Hercules,Calif.) in PBS containing 100 μg/mlBSA.

EXAMPLE 7

Detection of Recombinant FGF-20 by Western Blotting Analysis—The culturemedium or rat recombinant FGF-20 was separated by sodium dodecyl sulfate(SDS)-polyacrylamide gel (12.5%) electrophoresis under reducingconditions and transferred onto a nitrocellulose membrane (Hybond-ECL,Amersham, Buckinghamshire, England). The membrane was incubated withanti-E tag antibodies (1:500) (Pharmacia Biotech, Uppsala, Sweden). Theprotein with the E-tag was visualized as described (Hoshikawa, M. etal., Biochem. Biophys. Res. Commun. 244:187-191 (1998)).

Example 8

Rat Midbrain Cultured Cells—The ventral mesencephalon was resected fromrat embryos (E16.5). The mesencephalic blocks were washed 10 times withHanks' solution and mechanically dissociated without enzymatictreatment. The midbrain cultured cells were prepared essentially asdescribed (Sawada, H. et al., J. Neurosci. Res. 43:503-510 (1996)). Theculture medium consisted of Eagle's minimum essential medium (EMEM)supplemented with 0.2% sodium carbonate, 0.1% glucose, 0.029%L-glutamine and 0.238% HEPES. The cultured cells were incubated at 37°C. in the culture medium containing 10% fetal calf serum. From the5^(th) day of culture, the cells were incubated in the culture mediumcontaining 10% horse serum.

Example 9

Examination of Neurotrophic Activity of FGF-20 for Midbrain DopaminergicNeurons—Cells on the 8^(th) day of culture were incubated in Eagle'sminimum essential medium supplemented with 0.2% sodium hydrogencarbonate, 0.1% glucose, 0.029% L-glutamine, 0.238% HEPES and 10% horseserum or 0.1% bovine serum albumin in the presence or absence of FGF-20for 4 days and then fixed with fresh 4% paraformaldehyde for 30 min onthe 12^(th) day. Cells on the 8^(th) day of culture were also incubatedin the presence or absence of recombinant rat FGF-20 for 24 h and thenwere treated with 1 mM glutamate for 10 min. The cultured cells werefurther incubated in medium without FGF-20 and 1 mM glutamate and thenfixed with 4% paraformaldehyde for 30 min on the 12^(th) day. The fixedcells were washed with PBS for 15 min, and then treated with 0.2% TritonX-10 for 30 min. The cells were immunostained with anti-tyrosinehydroxylase (TH) antibody (Eugene Tech, Ridgefield Park, N.J.)essentially as described (Sawada, H. et al., J. Neurosci. Res.43:503-510 (1996)). Numbers of cultured dopaminergic neurons wereevaluated by counting cells stained with anti-TH antibody.

Example 10

Isolation and Analysis of Human FGF-20—The coding region of human FGF-20DNA was amplified from human brain cDNA library (λgt10) by PCR usingprimers specific for FGF-20 and λgt10 DNA. The nucleotide sequence ofthe cDNA encoding the carboxy-terminal 112 amino acids of human FGF-20was determined, and is shown in FIG. 7. An alignment of rat and humanFGF-20 amino acid sequences is shown in FIG. 8.

Example 11

Preparation of Antisera to FGF-20 by Immunization of Rabbits with anFGF-20 Peptide—A peptide sequence corresponding to selected contiguousamino acids of the human FGF-20 protein is synthesized and coupled tokeyhole limpet hemocyanin (KLH) as described (Harlow and Land,Antibodies: A Laboratory Manual, 1988. Cold Spring Harbor Laboratory,New York, N.Y.). The KLH-coupled peptide is used to immunize rabbits.Antisera are tested for specificity to FGF-20, and for cross-reactivitywith other FGF proteins.

Exemplary peptide sequences are: 1. RDGARSKRHQKFTH (SEQ ID NO:5) 2.QLAHLHGILRRRQLY (SEQ ID NO:6)

Example 12

Binding of FGF-20 to the Recombinant Extracellular Domains of FGFR-1c,FGFR-2c, and FGFR-3c—Recombinant FGF-20 was fixed on the sensor tip CM5(Amersham Pharmacia Biotech). Binding of the recombinant extracellulardomain of FGFR-1c, FGFR-2c, or FGFR-3c to FGF-20 on the tip was analyzedusing the BIACORE 2000 System (Amersham Pharmacia Biotech). Theequilibrium dissociation constant was determined by the BIA evaluationsoftware (Amersham Pharmacia Biotech). TABLE 1 BINDING OF FGF-20 TO FGFRECEPTORS Receptor K_(diss) (S⁻¹) K_(ass) (M⁻¹ · S⁻¹) K_(d) (M) FGFR-1cnd nd FGFR-2c 1.67 × 10⁻² 5.95 × 10⁵ 2.81 × 10⁻⁸ FGFR-3c 2.47 × 10⁻²1.15 × 10⁵ 2.17 × 10⁻⁷nd: not detected

As shown in Table 1, FGF-20 binds to FGF receptors 2 and 3, but not toFGF receptor 1. Thus, FGF-20 may exhibit biological effects not found inmembers of the FGF family that bind to FGF receptor 1, such as FGF-2 andFGF-4.

All patents, published patent applications and publications cited hereinare incorporated by reference as if set forth fully herein.

Although certain preferred embodiments have been described herein, it isnot intended that such embodiments be construed as limitations on thescope of the invention except as set forth in the following claims.

1-11. (canceled)
 12. An isolated polypeptide comprising amino acids atleast 95% identical to amino acids selected from the group consistingof: (a) amino acids from about 1 to about 211 of SEQ ID NO:4; (b) aminoacids from about 2 to about 211 of SEQ ID NO:4; (c) amino acids fromabout 170 to about 186 of SEQ ID NO:4; (d) amino acids from about 1 toabout 169 and from about 187 to about 211 of SEQ ID NO:4, wherein saidamino acids at positions about 169 and about 187 are joined by a peptidebond; and (e) amino acids from about 59 to about 193 of SEQ ID NO:4. 13.An isolated polypeptide wherein, except for at least one conservativeamino acid substitution, said polypeptide has an amino acid sequenceselected from the group consisting of: (a) amino acids from about 1 toabout 211 of SEQ ID NO:4; (b) amino acids from about 2 to about 211 ofSEQ ID NO:4; (c) amino acids from about 170 to about 186 of SEQ ID NO:4;(d) amino acids from about 1 to about 169 and from about 187 to about211 of SEQ ID NO:4, wherein said amino acids at positions about 169 andabout 187 are joined by a peptide bond; and (e) amino acids from about59 to about 193 of SEQ ID NO:4.
 14. An isolated polypeptide comprisingamino acids selected from the group consisting of: (a) amino acids fromabout 1 to about 211 of SEQ ID NO:4; (b) amino acids from about 2 toabout 211 of SEQ ID NO:4; (c) amino acids from about 170 to about 186 ofSEQ ID NO:4; (d) amino acids from about 1 to about 169 and from about187 to about 211 of SEQ ID NO:4, wherein said amino acids at positionsabout 169 and about 187 are joined by a peptide bond; and (e) aminoacids from about 59 to about 193 of SEQ ID NO:4.
 15. An epitope-bearingportion of the polypeptide of SEQ ID NO:4.
 16. The epitope-bearingportion of claim 15, which comprises between 10 and 50 contiguous aminoacids of SEQ ID NO:4.
 17. The epitope-bearing portion of claim 15, whichcomprises a polypeptide selected from the group consisting of aminoacids RDGARSKRHQKFTH (SEQ ID NO:5) and QLAHLHGILRRRQLY (SEQ ID NO:6).18. An isolated antibody that binds specifically to the polypeptide ofclaim
 12. 19. An isolated antibody that binds specifically to thepolypeptide of claim
 13. 20. An isolated antibody that bindsspecifically to the polypeptide of claim
 14. 21. A pharmaceuticalcomposition comprising the polypeptide of claim 12, in combination witha pharmaceutically acceptable carrier. 22-44. (canceled)
 45. An isolatedpolypeptide comprising amino acids at least 95% identical to amino acidsselected from the group consisting of: (a) amino acids from about 1 toabout 212 of SEQ ID NO:2; (b) amino acids from about 2 to about 212 ofSEQ ID NO:2; (c) amino acids from about 170 to about 186 of SEQ ID NO:2;(d) amino acids from about 1 to about 169 and from about 187 to about212 of SEQ ID NO:2, wherein said amino acids at positions about 169 andabout 187 are joined by a peptide bond; and (e) amino acids from about59 to about 193 of SEQ ID NO:2.
 46. An isolated polypeptide wherein,except for at least one conservative amino acid substitution, saidpolypeptide has an amino acid sequence selected from the groupconsisting of: (a) amino acids from about 1 to about 212 of SEQ ID NO:2;(b) amino acids from about 2 to about 212 of SEQ ID NO:2; (c) aminoacids from about 170 to about 186 of SEQ ID NO:2; (d) amino acids fromabout 1 to about 169 and from about 187 to about 212 of SEQ ID NO:2,wherein said amino acids at positions about 169 and about 187 are joinedby a peptide bond; and (e) amino acids from about 59 to about 193 of SEQID NO:2.
 47. An isolated polypeptide comprising amino acids selectedfrom the group consisting of: (a) amino acids from about 1 to about 212of SEQ ID NO:2; (b) amino acids from about 2 to about 212 of SEQ IDNO:2; (c) amino acids from about 170 to about 186 of SEQ ID NO:2; (d)amino acids from about 1 to about 169 and from about 187 to about 212 ofSEQ ID NO:2, wherein said amino acids at positions about 169 and about187 are joined by a peptide bond; and (e) amino acids from about 59 toabout 193 of SEQ ID NO:2.
 48. An epitope-bearing portion of thepolypeptide of SEQ ID NO:2.
 49. The epitope-bearing portion of claim 48,which comprises between 10 and 50 contiguous amino acids of SEQ ID NO:2.50. An isolated antibody that binds specifically to the polypeptide ofclaim
 45. 51. An isolated antibody that binds specifically to thepolypeptide of claim
 46. 52. An isolated antibody that bindsspecifically to the polypeptide of claim
 47. 53. A polypeptide encodedby a nucleic acid molecule comprising the polynucleotide of SEQ ID NO:1.54. The polypeptide of claim 53, comprising the amino acid sequence ofSEQ ID NO:2.
 55. A polypeptide encoded by a nucleic acid moleculecomprising the polynucleotide of SEQ ID NO:3.
 56. The polypeptide ofclaim 55, comprising the amino acid sequence of SEQ ID NO:4.